Should I choose Probe or Dye Based chemistry for my qPCR experiments?

When you are optimizing a new experiment and you need to test many primer pairs, it is usually best to start with TB Green based assays. TB Green enables you to find the most specific primers without the need to design and purchase several probes. TB Green also allows melting curve analysis, which can reveal undesirable primer-dimers or other nonspecific PCR products. Probe or TaqMan chemistries [...]

What are the differences between absolute and relative quantification?

*Absolute quantification determines the absolute number (i.e., number of copies) of a target using a standard sample that has a known number of copies. *Relative quantifications allow a relative comparison between samples. Typically, using relative quantification, a target gene to be quantified and a reference gene (e.g., a housekeeping gene) are simultaneously assayed for the purpose of [...]

I understand that there are two ways to determine the Ct value. What are they?

The Ct value can be determined by two different methods: 1. The crossing point method defines the Ct value as the crossing point between the amplification curve and the threshold line. 2. The second derivative maximum method defines the Ct value as the point of the maximum of the second derivative of amplification curve (second differential curve). The latter method allows highly precise [...]

What is the sensitivity of qPCR?

The sensitivity of qPCR varies depending on experimental conditions such as the reagents and primers used. Appropriately designed systems have demonstrated the ability to detect as few as ~10 copies of template.Real-time PCR [...]

What is the appropriate number of replicates (n)?

The necessary number of sample replicates (n) varies depending on the experimental system. In principle, when the experimental error is expected to be relatively large, use a larger number of samples. *When profiling gene expression by RT-qPCR, it is useful to prepare multiple biological replicates and perform multiple RNA extractions in order to ascertain the degree of biological variability. [...]